A native antibody molecule consists of two identical heavy chains, and two identical light chains. The heavy chain constant region includes CH1, the hinge region, CH2, and CH3. Papain digestion of antibodies produces two fragments, Fab and Fc. The Fc fragment consists of CH2, CH3, and part of the hinge region. In human IgG molecules, the Fc fragment is generated by papain cleavage of the hinge region N-terminal to Cys 226. Therefore, the human IgG heavy chain Fc region is usually defined as stretching from the amino acid residue at position 226 to the C-terminus (numbered according to the EU index of Kabat et al., “Sequences of Proteins of Immunological Interest”, 5th ed., National Institutes of Health, Bethesda, Md. (1991); the EU numbering scheme is used hereinafter).
It has been recognized that the Fc region is critical for maintaining the serum half-life of an antibody of class IgG (Ward and Ghetie, Ther. Immunol. 2:77–94 (1995)). Studies have found that the serum half-life of an IgG antibody is mediated by binding of Fc to the neonatal Fc receptor (FcRn). FcRn is a heterodimer consisting of a transmembrane a chain and a soluble β chain (β2-microglobulin). FcRn shares 22–29% sequence identity with Class I MHC molecules and has a non-functional version of the MHC peptide-binding groove (Simister and Mostov, Nature 337:184–187 (1989)). The α1 and α2 domains of FcRn interact with the CH2 and CH3 domains of the Fc region (Raghavan et al., Immunity 1:303–315 (1994)).
A model has been proposed for how FcRn might regulate the serum half-life of an antibody. According to this model, IgGs are taken up by endothelial cells through non-specific pinocytosis and then enter acidic endosomes. FcRn binds IgG at acidic pH (<6.5) in endosomes and releases IgG at basic pH (>7.4) in the bloodstream. Accordingly, FcRn salvages IgG from a lysosomal degradation pathway. When serum IgG levels decrease, more FcRn molecules are available for IgG binding so that an increased amount of IgG is salvaged. Conversely, if serum IgG levels rise, FcRn becomes saturated, thereby increasing the proportion of pinocytosed IgG that is degraded (Ghetie and Ward, Annu. Rev. Immunol. 18:739–766 (2000)).
Consistent with the above model, the results of numerous studies support a correlation between the affinity for FcRn binding and the serum half-life of an antibody (Ghetie and Ward, ibid.). Significantly, such a correlation has been extended to engineered antibodies with higher affinity for FcRn than their wild-type parent molecules. A large number of publications and patents based upon mutagenesis studies support this correlation (see e.g., Ghetie et al., Nat. Biotechnol. 15:637–640 (1997); Shields et al., J. Biol. Chem. 276:6591–6604 (2001); Dall'Acqua et al., J. Immunol. 169:5171–5180 (2002); Hinton et al., J. Biol. Chem. 279:6213–6216 (2004); Kim et al., Eur. J. Immunol. 29:2819–2825 (1999); Homick et al., J. Nucl. Med. 41:355–362 (2000); U.S. Pat. No. 6,165,745; U.S. Pat. No. 6,277,375 B1; U.S. Patent Application Publication No. 20020098193; PCT Publication WO 97/34621; and PCT Publication WO 02/060919). In addition, PCT Publication No. WO 98/05787 discloses deleting or substituting amino acids at positions 310–331 of the BR96 antibody in order to reduce its induced toxicity.
U.S. patent application Ser. No. 10/687,118, filed Oct. 15, 2003 (and hereby incorporated herein by reference in its entirety) and corresponding PCT Publication No. WO 04/035752 discloses mutations at positions 250, 314, and 428 of the Fc heavy chain constant region that provide modified antibodies with altered FcRn binding affinity and/or serum half-life relative to unmodified antibody.
Advances in molecular biology techniques have allowed the preparation of novel chimeric polypeptides with multiple functional domains. The most common of such chimeric polypeptides are immunoglobulin (Ig) fusion proteins. These proteins consist of the Fc regions of antibodies, typically mouse or human antibodies, fused to an unrelated protein or protein fragment. Such Fc-fusion proteins are valuable for studying protein function in vitro and in vivo and have potential therapeutic and diagnostic use in the clinical setting.
Methods for fusing or conjugating polypeptides to the constant regions of antibodies (i.e. making Fc fusion proteins) are described in, e.g., U.S. Pat. Nos. 5,336,603, 5,622,929, 5,359,046, 5,349,053, 5,447,851, 5,723,125, 5,783,181, 5,908,626, 5,844,095, and 5,112,946; EP 307,434; EP 367,166; EP 394,827; PCT publications WO 91/06570, WO 96/04388, WO 96/22024; WO 97/34631, and WO 99/04813; Ashkenazi et al., Proc. Natl. Acad. Sci. USA 88:10535–10539 (1991); Traunecker et al., Nature 331:84–86 (1988); Zheng et al., J. Immunol. 154:5590–5600 (1995); and Vil et al., Proc. Natl. Acad. Sci. USA 89:11337–11341 (1992), which are incorporated herein by reference in their entireties.